PROJECT SUMMARY The ability of stem cells to survive and maintain function during tissue turnover and stress is a key determinant of long-term regenerative success. Skeletal muscle stem cells lose fitness under contexts of high stress including aging, muscular disease and irradiation (IR). Hence there is a critical need to identify the properties that maintain or enhance SC stress resistance. During the previous funding cycle, we demonstrated that self- renewal potential was restricted to slowly dividing SCs (based on label retention). Our long-term goal is to understand SC heterogeneity and identify the molecular regulators that govern SC potency. We have identified Pax3 as a discrete marker of stress-resistant SCs. These cells are rare in number, express typical satellite cell markers, proliferate slowly, as indicated by label retention analysis, and are capable of self-renewal and differentiation. Under conditions of stress this subset is preserved and becomes the dominant source of stem cells for muscle repair and stem cell replenishment after injury. This is the first demonstration of clonal expansion of a discrete muscle stem cell subset in vivo. While Pax3 has been studied in development, its role in stress resistance of quiescent adult SCs is completely unknown. The overall goal of this proposed study is therefore to understand the functional capacity and regulation of a specific subset of stress-resistant SCs. The first aim will combine lineage tracing (Tmx-inducible Pax3CreERT2.ROSA26-nTNG) and cell ablation (DTAfl/fl) strategies to determine the cellular contribution and requirement of adult Pax3+ SC lineage to muscle repair during normal injury or after increased stress (IR and transplantation), to ask whether Pax3+ SCs fulfill the definition of a reserve stem cell. The second aim will determine the dynamics between pax3 expression (pax3gfp+/- knockin allele) or proliferative output (TetO-H2B-mCHerry) in the progeny of Pax3+ and Pax3- SC lineages (Pax3CreERT2.ROSA26-reporter) during muscle tissue turnover. The third aim will dissect the molecular mechanism underpinning Pax3+ SC function. We will examine the requirement and sufficiency of Pax3 in SC function using loss and gain of function approaches and its regulation through cell survival/apoptosis machinery. Understanding the requirement and regulation of Pax3 in adult SCs will provide new insights into adult SC biology independent of Aim 1 and 2. Successful completion of the proposed aims will provide evidence for a pre-determined subset of Pax3+ SCs, and reveal the clonal dynamics among muscle stem cells that manifests under the selective pressure of stress. Understanding the potency and regulation of stress resistant SCs will lead to important insights for cell therapy, patients undergoing radiation therapy and other physiological stressors including degenerative diseases and aging.